Impact on the clinical evolution of patients with COVID-19 from an Intensive Care Unit with isolation of Candida spp. in respiratory samples / Impacto na evolução clínica de pacientes com COVID-19 de uma Unidade de Terapia Intensiva com isolamento de Candida spp. em amostras respiratórias / Impacto en la evolución clínica de pacientes con COVID-19 de una Unidad de Cuidados Intensivos con aislamiento de Candida spp. en muestras respiratorias

Background and Objectives: several patients with COVID-19 require hospital admission due to severe respiratory complications and undergo intensive care with mechanical ventilation (MV) support. Associated with this situation, there is an increase in fungal co-infections, which has a negative impact on the outcome of COVID-19. In this regard, this study intended to compare Candida spp. incidence in the respiratory tract of patients admitted in the COVID and General Intensive Care Units (ICU) at a teaching hospital in 2021. Methods: the results of protected tracheal aspirate samples from 556 patients admitted to the COVID ICU and 260 to General ICU as well as the respective records. Results: of the patients analyzed, 38 revealed a positive sample for Candida in the COVID ICU and 10 in the General ICU, with an incidence of 68.3/1000 and 38.5/1000, respectively. Males were predominant in both wards. The most affected age group was the population over 60 years old, and the average hospital admission for the COVID ICU was 22.1 years, and for the General ICU, 24.2. Conclusion: Candida albicans was the most frequently isolated species, and the mortality rate in patients positive for Candida was higher in patients with COVID-19 compared to patients in the General ICU, suggesting that patients infected with SARS-CoV-2, admitted to the ICU under MV, are more predisposed to colonization by Candida spp., which can have a fatal outcome in these patients.(AU)

Justificativa e objetivos: muitos pacientes com COVID-19 necessitam de hospitalização devido às complicações respiratórias graves, e são submetidos a cuidados intensivos com suporte de ventilação mecânica (VM). Associado a esse quadro, verifica-se o aumento de coinfecções fúngicas, que tem impacto negativo no desfecho da COVID-19. Nesse sentido, este estudo pretendeu comparar a incidência de Candida spp. no trato respiratório de pacientes internados nas Unidades de Terapia Intensiva (UTI) COVID e Geral em um hospital escola em 2021. Métodos: foram avaliados os resultados de amostras de aspirado traqueal protegido provenientes de 556 pacientes internados na UTI COVID e 260 na UTI Geral, bem como os respectivos prontuários. Resultados: dos pacientes analisados, 38 revelaram amostra positiva para Candida na UTI COVID e 10 na UTI Geral, com incidência de 68,3/1000 e 38,5/1000, respectivamente. O sexo masculino foi predominante em ambas as alas. A faixa etária mais acometida foi a população acima de 60 anos, e a média de internação para a UTI COVID foi de 22,1 anos, e para a UTI Geral, 24,2. Conclusão: Candida albicans foi a espécie isolada com maior frequência, e a taxa de mortalidade em pacientes com positivos para Candida foi maior em pacientes com COVID-19 em relação aos pacientes da UTI Geral, sugerindo que pacientes infectados com SARS-CoV-2, internados em UTI sob VM, são mais predispostos à colonização por Candida spp., que pode ter um desfecho fatal nesses pacientes.(AU)

Justificación y objetivos: muchos pacientes con COVID-19 requieren hospitalización debido a complicaciones respiratorias graves y se someten a cuidados intensivos con soporte de ventilación mecánica (VM). Asociado a esta situación, hay un aumento de las coinfecciones fúngicas, lo que repercute negativamente en el desenlace de la COVID-19. En este sentido, este estudio pretendió comparar la incidencia de Candida spp. en el tracto respiratorio de pacientes ingresados en las Unidades de Cuidados Intensivos (UCI) COVID y General de un hospital escuela en 2021. Métodos: los resultados de muestras de aspirado traqueal protegidas de 556 pacientes ingresados en la UCI COVID y 260 en el UCI General, así como los respectivos registros. Resultados: de los pacientes analizados, 38 presentaron muestra positiva a Candida en UCI COVID y 10 en UCI General, con una incidencia de 68,3/1000 y 38,5/1000, respectivamente. Los machos predominaban en ambas alas. El grupo de edad más afectado fue la población mayor de 60 años, y la hospitalización promedio en la UCI COVID fue de 22,1 años, y en la UCI General, de 24,2. Conclusiones: Candida albicans fue la especie aislada con mayor frecuencia, y la tasa de mortalidad en pacientes positivos para Candida fue mayor en pacientes con COVID-19 en comparación con los pacientes en la UCI General, lo que sugiere que los pacientes infectados con SARS-CoV-2, ingresados en la UCI bajo VM, están más predispuestos a la colonización por Candida spp., lo que puede tener un desenlace fatal en estos pacientes.(AU)

The Response of Paracoccidioides lutzii to the Interaction with Human Neutrophils.

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

Evaluation of Five Non-Culture-Based Methods for the Diagnosis of Meningeal Sporotrichosis.

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture's limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

Murine macrophages do not support the proliferation of Leishmania (Viannia) braziliensis amastigotes even in absence of nitric oxide and presence of high arginase activity.

Leishmania (Viannia) braziliensis is the main species responsible for American tegumentary leishmaniasis in Brazil. Nevertheless, the use of this parasite species to study Leishmania infection in the murine model has been less conducted when compared with other Leishmania species. The control of murine infection with Leishmania has been associated with nitric oxide (NO) produced by inducible NO synthase (iNOS) from M1 macrophages, while arginase expressed by M2 macrophages is related to Leishmania proliferation. Here we use three different strains of L. (V.) braziliensis and one strain of L. (L.) major to study a 9-day infection of macrophages in vitro. Wild-type bone marrow-derived macrophages (BMDM) supported the proliferation of L. (L) major amastigotes from the 3rd day after infection, while all strains of L. (V.) braziliensis did not proliferate even inside IL-4-treated or iNOS knockout (KO) macrophages. The arginase activity was higher in iNOS KO than IL-4-treated macrophage showing that the absence of proliferation is independent of arginase. Importantly, L. (V.) braziliensis was able to cause uncontrolled disease in iNOS KO mice in vivo demonstrating that murine macrophages present at the site of infection have additional changes beyond inhibition of NO production or stimulation of arginase activity to support parasite proliferation.

Comparative Proteomic Analysis of Histoplasma capsulatum Yeast and Mycelium Reveals Differential Metabolic Shifts and Cell Wall Remodeling Processes in the Different Morphotypes.

Histoplasma capsulatum is a thermally dimorphic fungus distributed worldwide, but with the highest incidence in the Americas within specific geographic areas, such as the Mississippi River Valley and regions in Latin America. This fungus is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and is related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have made important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, H. capsulatum proteome studies have been underexplored. In the present study, we report the first proteomic comparison between the mycelium and the yeast cells of H. capsulatum. Liquid chromatography coupled to mass spectrometry was used to evaluate the proteomic profile of the two phases of H. capsulatum growth, mycelium, and yeast. In summary, 214 and 225 proteins were only detected/or preferentially abundant in mycelium or yeast cells, respectively. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation occurred in greater abundance, suggesting a higher use of anaerobic pathways for energy production. In yeast cells, proteins related to the tricarboxylic acid cycle and response to temperature stress were in high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Proteomic data validation was performed by enzymatic activity determination, Western blot assays, or immunofluorescence microscopy. These experiments corroborated, directly or indirectly, the abundance of isocitrate lyase, 2-methylcitrate synthase, catalase B, and mannosyl-oligosaccharide-1,2-alpha-mannosidase in the mycelium and heat shock protein (HSP) 30, HSP60, glucosamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-phosphate mutase in yeast cells. The proteomic profile-associated functional classification analyses of proteins provided new and interesting information regarding the differences in metabolism between the two distinct growth forms of H. capsulatum.

Interacting with Hemoglobin: Paracoccidioides spp. Recruits hsp30 on Its Cell Surface for Enhanced Ability to Use This Iron Source.

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.

Insights Into Histoplasma capsulatum Behavior on Zinc Deprivation.

Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.

Beyond Melanin: Proteomics Reveals Virulence-Related Proteins in Paracoccidioides brasiliensis and Paracoccidioides lutzii Yeast Cells Grown in the Presence of L-Dihydroxyphenylalanine.

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen's survival in the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was induced using L-dihydroxyphenylalanine as a precursor, and quantitative proteomics were performed using reversed-phase nano-chromatography coupled to high-resolution mass spectrometry. When comparing melanized versus non-melanized cells, 1006 and 582 differentially abundant/detected proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 important KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways in melanized P. brasiliensis and 18 in P. lutzii, while differentially abundant proteins from non-melanized cells from these species were involved in 21 and 25 enriched pathways, respectively. Melanized cells presented an abundance of additional virulence-associated proteins, such as phospholipase, proteases, superoxide dis-mutases, heat-shock proteins, adhesins, and proteins related to vesicular transport. The results suggest that L-dihydroxyphenylalanine increases the virulence of Paracoccidioides spp. through complex mechanisms involving not only melanin but other virulence factors as well.

Efeito do extrato da própolis e do digluconato de clorexidina sobre a formação de biofilme por Candida albicans em resina acrílica / Effect of propolis extract and chlorhexidine digluconate on the biofilm formation by Candida albicans in acrylic resin

Objetivo: avaliar in vitro a ação antimicrobiana do extrato da própolis (EP) e do digluconato de clorexidina (DCHX) na formação de biofilme por Candida albicans em resina acrílica termopolimerizada. Métodos: o efeito do EP e DCHX em biofilmes de C. albicans foi avaliado pela quantificação de Unidades Formadoras de Colônias (UFCs), pela quantificação da biomassa por cristal violeta e de polissacarídeos por safranina. Para tanto, C. albicans foram pré-aderidas em corpos de prova e somente em microplacas de poliestireno, posteriormente foi realizado o tratamento com diferentes concentrações de EP (221 µg/mL e 443 µg/mL) e DCHX (0,25% e 0,5%). Resultados: foi demonstrada uma redução significativa na formação de biofilme por C. albicans com ambas as substâncias testadas e em todas as concentrações. Conclusão: de acordo com os resultados, observou-se que ambos os tratamentos foram eficazes na redução do biofilme e que o EP, por ser um produto natural, de baixo custo e sem afeitos colaterais, representa uma alternativa inovadora para o tratamento da candidose oral em usuários de próteses removíveis.(AU)

Objective: to evaluate in vitro the antimicrobial action of propolis extract (EP) and chlorhexidine digluconate (DCHX) in the biofilm formation by Candida albicans in thermopolymerized acrylic resin. Methods: the effect of EP and DCHX on C. albicans biofilms were evaluated through the quantification of Colony Forming Units (CFU's), the quantification of biomass by violet crystal and polysaccharides by safranin. For this purpose, C. albicans were pre-adhered to specimens and only on polystyrene microplates and subsequently the treatment was performed with different concentrations of EP (221 µg/mL and 443 µg/mL) and DCHX (0.25% and 0,5%). Results: a significant reduction in the biofilm formation by C. albicans was demonstrated, with both substances tested and in all concentrations. Conclusion: according to the results, it was observed that both treatments were effective in reducing biofilm and EP, being a natural product, low cost and without side effects, represents an innovative alternative for the treatment of oral candidiasis in users removable dentures.(AU)

Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells.

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

Interactome of Glyceraldehyde-3-Phosphate Dehydrogenase Points to the Existence of Metabolons in Paracoccidioides lutzii.

Paracoccidioides is a dimorphic fungus, the causative agent of paracoccidioidomycosis. The disease is endemic within Latin America and prevalent in Brazil. The treatment is based on azoles, sulfonamides and amphotericin B. The seeking for new treatment approaches is a real necessity for neglected infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential glycolytic enzyme, well known for its multitude of functions within cells, therefore categorized as a moonlight protein. To our knowledge, this is the first approach performed on the Paracoccidioides genus regarding the description of PPIs having GAPDH as a target. Here, we show an overview of experimental GAPDH interactome in different phases of Paracoccidioides lutzii and an in silico analysis of 18 proteins partners. GAPDH interacted with 207 proteins in P. lutzii. Several proteins bound to GAPDH in mycelium, transition and yeast phases are common to important pathways such as glycolysis and TCA. We performed a co-immunoprecipitation assay to validate the complex formed by GAPDH with triose phosphate isomerase, enolase, isocitrate lyase and 2-methylcitrate synthase. We found GAPDH participating in complexes with proteins of specific pathways, indicating the existence of a glycolytic and a TCA metabolon in P. lutzii. GAPDH interacted with several proteins that undergoes regulation by nitrosylation. In addition, we modeled the GAPDH 3-D structure, performed molecular dynamics and molecular docking in order to identify the interacting interface between GAPDH and the interacting proteins. Despite the large number of interacting proteins, GAPDH has only four main regions of contact with interacting proteins, reflecting its ancestrality and conservation over evolution.

Metabolic Peculiarities of Paracoccidioides brasiliensis Dimorphism as Demonstrated by iTRAQ Labeling Proteomics.

Paracoccidioidomycosis (PCM), a systemic mycosis with a high incidence in Latin America, is caused by thermodimorphic fungi of the Paracoccidioides genus. The contact with host occurs by the inhalation of conidia or mycelial propagules which once reaching the pulmonary alveoli differentiate into yeast cells. This transition process is vital in the pathogenesis of PCM allowing the fungus survival in the host. Thus, the present work performed a comparative proteome analysis of mycelia, mycelia-to-yeast transition, and yeast cells of Paracoccidioides brasiliensis. For that, tryptic peptides were labeled with iTRAQ and identified by LC-MS/MS and computational data analysis, which allowed the identification of 312 proteins differentially expressed in different morphological stages. Data showed that P. brasiliensis yeast cells preferentially employ aerobic beta-oxidation and the tricarboxylic acid cycle accompanied by oxidative phosphorylation for ATP production, in comparison to mycelia and the transition from mycelia-to-yeast cells. Furthermore, yeast cells show a metabolic reprogramming in amino acid metabolism and in the induction of virulence determinants and heat shock proteins allowing adaptation to environmental conditions during the increase of the temperature. In opposite of that, the alcoholic fermentation found to P. lutzii, at least under laboratory conditions, is strongly favored in mycelium compared to yeast cells. Thereby, the data strongly support substantial metabolic differences among members of the Paracoccidioides complex, when comparing the saprobiotic mycelia and the yeast parasitic phases.

Identification and characterization of Paracoccidioides lutzii proteins interacting with macrophages.

Paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a systemic disorder that involves the lungs and other organs. The adherence of pathogenic microorganisms to host tissues is an essential event in the onset of colonization and spread. The host-pathogen interaction is a complex interplay between the defense mechanisms of the host and the efforts of pathogenic microorganisms to colonize it. Therefore, the identification of fungi proteins interacting with host proteins is an important step understanding the survival strategies of the fungus within the host. In this paper, we used affinity chromatography based on surface proteomics (ACSP) to investigate the interactions of pathogen proteins with host surface molecules. Paracoccidioides lutzii extracts enriched of surface proteins were captured by chromatographic resin, which was immobilized with macrophage cell surface proteins, and identified by mass spectrometry. A total of 215 proteins of P. lutzii were identified interacting with macrophage proteins. In silico analysis classified those proteins according to the presence of sites for N- and O-glycosylation and secretion by classical and non-classical pathways. Serine proteinase (SP) and fructose-1,6-bisphosphate aldolase (FBA) were identified in our proteomics analysis. Immunolocalization assay and flow cytometry both showed an increase in the expression of these two proteins during host-pathogen interaction.

Epidemiological investigation and molecular typing of dermatophytosis caused by Microsporum canis in dogs and cats.

The objective of the present study was investigate the prevalence of dermatophytes in dogs, cats and environment floor through molecular epidemiology tools to identify the genetic profile of these infectious agents. This was an observational study with cross-sectional surveys design. Sample were collected from the hair and skin of 52 dogs and cats with the clinical suspicion of dermatophytosis, over a period of one year in Maringá, in the state of Paraná, Brazil. Household samples (carpets and floor), were collected from animals that were positive for dermatophytosis by morphological colonies characteristics, and samples of dogs or cats living in the same household as with the positive animals were also collected. After mycological confirmation, molecular typing was performed by random amplified polymorphic DNA (RAPD). Microsporum canis was the unic dermatophyto isolated whose prevalence was 26.9% (14/52) in animals with the clinical suspicion of dermatophytosis and four other animals that lived with positive animals. As some animals had more than one lesion site, there were 22 total positive cultures from samples from animals and another ten from abiotic sources. The majority of the animals that provided positive cultures for M. canis were aged up to five months (77.8%) and were female (66.7%). Molecular typing using the P1 primer revealed genetically distinct profiles in the symptomatic, asymptomatic and environmental animal samples, or the same animal, furthermore, showed that M. canis could have microevolution.

Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma.

Although members of the Paracoccidioides complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MS E technology, we analyzed the proteomic response of Paracoccidioides brasiliensis during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of P. brasiliensis. The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite-host relationship and pathogenesis of PCM.

A proteomic dataset of secreted proteins by three Staphylococcus saprophyticus strains.

This article presents a proteomic dataset generated from a comparative analysis of the exoproteome of Staphylococcus saprophyticus, ATCC 15305, 7108 and 9325 strains. The extract of secreted proteins were obtained after incubation of stationary phase cells in BHI medium. All samples were submitted to nano-ESI-UPLC-MSE, and the spectrum obtained was processed and analyzed by ProteinLynx Global Server (PLGS), Uniprot and Pedant databases, for identification, annotation and functional classification of proteins. Fold changes and protein relative abundances were properly reported. This report is related to the research article entitled "The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents" (Oliveira et al., 2018). The proteomic data generated have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with a project number PXD008643, https://www.ebi.ac.uk/pride/archive/projects/PXD008643.

The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents.

Staphylococcus saprophyticus is a gram-positive microorganism responsible for urinary tract infections (UTIs). Although some virulence factors are characterized, such as urease, autolysins, adhesins and hemagglutinins, large-scale proteomic studies have not been performed within this species. We performed the characterization of the exoproteome from three S. saprophyticus strains: the reference strain ATCC 15,305, a non-capsular strain 7108 and the 9325 strain containing a thick capsule which were cultured in BHI medium and culture supernatants were analysed by using mass spectrometry approach. We observed a core of 72 secreted proteins. In addition, it was possible to detect diversity in the protein profiles of the exoproteomes. Interestingly, strain 7108 presented no secretion of three antigenic proteins, including the classical SsaA antigen. In addition, the level of antigenic proteins secreted by strain 9325 was higher than in ATCC 15,305. This result was confirmed by Western blot analysis using anti-SsaA polyclonal antibodies, and no production/ secretion of SsaA was detected in strain 7108. Transcriptional data shows that 7108 strain produces transcripts encoding SsaA, suggesting post-transcriptional regulation occurs in this strain. Moreover, when compared with the other strains that were analyzed, it was possible to detect higher levels of proteases secreted by strain 7108 and higher levels of antigenic proteins and transglycosylases secreted by 9325 strain. The results reveal diversity in protein secretion among strains. This research is an important first step towards understanding the variability in S. saprophyticus exoproteome profile and could be significant in explaining differences among strains.

Argentilactone Molecular Targets in Paracoccidioides brasiliensis Identified by Chemoproteomics.

Paracoccidioidomycosis (PCM) is the cause of many deaths from systemic mycoses. The etiological agents of PCM belong to the Paracoccidioides genus, which is restricted to Latin America. The infection is acquired through the inhalation of conidia that primarily lodge in the lungs and may disseminate to other organs and tissues. The treatment for PCM is commonly performed via the administration of antifungals such as amphotericin B, co-trimoxazole, and itraconazole. The antifungal toxicity and side effects, in addition to their long treatment times, have stimulated research for new bioactive compounds. Argentilactone is a compound that was isolated from the Brazilian savanna plant Hyptis ovalifolia, and it has been suggested to be a potent antifungal, inhibiting the dimorphism of P. brasiliensis and the enzymatic activity of isocitrate lyase, a key enzyme of the glyoxylate cycle. This work was developed due to the importance of elucidating the putative mode of action of argentilactone. The chemoproteomics approach via affinity chromatography was the methodology used to explore the interactions between P. brasiliensis proteins and argentilactone. A total of 109 proteins were identified and classified functionally. The most representative functional categories were related to amino acid metabolism, energy, and detoxification. Argentilactone inhibited the enzymatic activity of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase. Furthermore, argentilactone induces the production of reactive oxygen species and inhibits the biosynthesis of cell wall polymers.

Characterization of extracellular proteins in members of the Paracoccidioides complex.

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) - an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MSE) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes.

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies.

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.